This proposal deals with further studies on development of in vitro culture systems able to produce the various life cycle stages o salivarian trypanosomes (Trypanosoma brucei sspp. and T. congolense) and to ensure that the forms grown in vitro resemble those found in mammalian hosts and tsetse flies infected with the same stock. The methods will be applied to trypanosome stocks recently isolated from well-separated areas of Africa. Cultures of bloodstream forms will be initiated from infected mouse blood or with metabyclic trypanosomes harvested from infective cultures maintained at 28 degrees C. Trypanosomes from these cultures or from infected mice will serve as a starting material for in vitro production of the vector stages. Special attention will be given to cultivation of metacyclic forms now available (in the case of T. brucei sspp.) from tsetse fly and Phormia tissue cultures. Suggested methods, some already attempted with some success are-- (a) further use of insect and mammalian tissues and cell lines incubated at 28 degrees C., (b) use of medium formulated from the chemical composition of tsetse fly saliva, and (c) pretreatment of the culture flasks with compounds, eg. collagen, known to enhance attachment of the epimastigote stage to the bottom of the flask and thus enable their transformation into metacylic forms. The structure and antigenicity of the stages of trypanosomes grown in vitro will be compared with these of the same stock occurring in vivo using light and electron microscopy as well as immunoflourescence and monoclonal antibody techics. Populations of metacyclic trypanosomes which develop in tsetse flies fed with, or in cultures initiated with organisms derived from cloned bloodstream forms usually contain several different variant antigenic types (VATs). It is planned to investigate if metacyclic trypanosomes produced from populations of cloned procyclic forms would be antigenically heterogeneous. There is still a need for additional methods to cultivate infective forms of salivarian trypanosomes which could be useful in immuno- and chemotherapy. A preliminary test on the use of an altruistic' vaccine will be made using the culture procyclic forms.